Synthesis and Biological Evaluation of Tylophorine-Derived

Article pubs.acs.org/jmc

Synthesis and Biological Evaluation of Tylophorine-Derived Dibenzoquinolines as Orally Active Agents: Exploration of the Role of Tylophorine E Ring on Biological Activity Yue-Zhi Lee,† Cheng-Wei Yang,†,‡ Hsing-Yu Hsu,† Ya-Qi Qiu,†,‡ Teng-Kuang Yeh,† Hsin-Yu Chang,† Yu-Sheng Chao,† and Shiow-Ju Lee*,† †

Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, No. 35 Keyan Road, Zhunan Town, Miaoli County 350, Taiwan ‡ Graduate Program of Biotechnology in Medicine, Institute of Molecular Medicine, National Tsing Hua University, Hsinchu, Taiwan S Supporting Information *

ABSTRACT: A series of novel tylophorine-derived dibenzoquinolines has been synthesized and their biological activity evaluated. Three assays were conducted: inhibition of cancer cell proliferation, inhibition of TGEV replication for anticoronavirus activity, and suppression of nitric oxide production in RAW264.7 cells (a measure of anti-inflammation). The most potent compound from these assays, dibenzoquinoline 33b, showed improved solubility compared to tylophorine 9a, in vivo efficacies in a lung A549 xenografted tumor mouse model and a murine paw edema model, good bioavailability, and no significant neurotoxicity (as tested by a rota-rod test for motor coordination). This is the first study to explore in detail the role of the tylophorine E ring on biological activity and very strongly suggests that tylophorine-derived dibenzoquinolines merit further development into orally active agents.

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INTRODUCTION Tylophorine is a phenanthroindolizidine alkaloid found in various herbs, some of which, e.g., Tylophora indica, Tylophora atrofolliculata, and Tylophora ovata, are used in the traditional medicines of several countries.1−3 Although their direct molecular targets are not known,2,4 they have been found to impart a multitude of biological activities, including anticancer,5−7 anti-inflammation,5,6 and anticoronavirus.8 Cancer cell toxicity is accomplished by interfering in the cell cycle and inhibiting the signaling of the transcriptional factors NF-κB and AP1.5,7 Their anti-inflammatory activity was found to arise through enhanced phosphorylation of Akt and down-regulation of AP1, thereby suppressing nitric oxide production in LPS/ IFNγ stimulated RAW264.7 cells.5 In TGEV (transmissible gastroenteritis coronavirus) infected ST cells, they inhibit coronavirus induced apoptosis and viral replication.8 In addition to the conventional pentacyclic phenanthroindolizidines and phenanthroquinolizidines, the biological activity of nonpentacyclic tylophorine derivatives has also been explored. © XXXX American Chemical Society

One class of tylophorine derivatives, the tyloindicine I analogue 7-(4-methoxyphenyl)-6-phenyl-2,3,8,8a-tetrahydroindolizin5(1H)-one (4) was proposed to operate through an unknown, novel mechanism of action.9 Another class of tylophorine derivatives, the phenanthrene-based 9-N replaced compounds, e.g., 5, was proposed to exert anticancer activity through a different mode of action by inactivation of Akt and inhibition of the NF-κB pathway signaling.10 The direct biological targets of these molecules, however, remain to be elucidated. Furthermore, although they all exert anticancer activity, their pharmacophores (for the molecular recognition by biological macromolecules) are likely differentiated, leading to changes in targeted molecules or mechanism of action. Development of tylophorine-related novel compounds, e.g., seco-tylophorine compounds9,11 and phenanthrene-based 9-N replaced compounds12,13 (Figure 1), into cytotoxic agents for Received: May 21, 2012

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dx.doi.org/10.1021/jm300705j | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Figure 1. Tylophorine and derived compounds.

Scheme 1. Synthesis of Tylophorine-Derived Dibenzoquinolines with Varied N-Substitutesa

a Reagents and conditions: (a) R-Br, NaH, DMF; (b) (i) NaI, CH3CN, 130 °C, overnight, (ii) Bu3SnH, AIBN, toluene, reflux, 6 h; (c) NaAl(OCH2CH2OMe)2H2, dioxane, 120 °C, 2 h; (d) LiAlH4, AlCl3, THF, rt, 4 h; (e) LiAlH4, THF, rt, 48 h, quench, MeOH/H2O (100:1), rt; (f) PPTS, MeOH, 55 °C, 20 h; (g) H2SO4, CH2Cl2, rt, 20 h.

series of derivatives bearing modifications at the E ring and/or differing N-substitutions. All derivatives were submitted for a variety of tests: anticell growth against a panel of cancer cell lines, antiviral replication in TGEV infected ST cells detected by inhibition of TGEV N and S protein expression, and suppression of nitric oxide production in LPS/IFNγ stimulated RAW264.7 cells. The counterpart C13a atom of the E ringuncyclized derivatives, dibenzoquinolines, is not a stereocenter. Several potent derivatives possessing the same biological activities as tylophorine in terms of anticancer cell proliferation, anti-TGEV activity, and anti-inflammation were discovered and their structure and activity relationships analyzed. Of them, dibenzoquinoline 33b exhibited improved solubility compared to tylophorine 9a, potent in vivo efficacy, and good bioavailability as an orally active agent without neurotoxicity.

cancer treatment faces formidable challenges. For example, the solubility of tylophorine in water is very poor. Also, the stereochemistry of these compounds affects their potency9,14 and thus isolation of enantiopure samples is needed to clarify structure−activity relationships and further development. Furthermore, a clinical trial of tylocrebrine in the 1960s failed due to the central nervous system (CNS) side effects.15 Increasing the polarity of tylophorine and derived compounds should prevent them from crossing the blood−brain barrier, thereby lowering CNS toxicity. Thus, the solubility, polarity, pharmacokinetic properties, oral availability, neurotoxicity, and synthetic routes toward these compounds must all be improved. Herein, we investigate the role of the tylophorine E ring (Figure 1) on biological activity through the synthesis of a B



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Scheme 2. Synthesis of Tylophorine-Derived Dibenzoquinolines with Varied Phenanthrene Substitutesa

Reagents and conditions: (a) Et3N, Ac2O, 90 °C, 15 h; (b) FeSO4·7H2O, NH4OH, 100 °C, 2 h; (c) i-C5H11ONO, H2SO4, NaI, acetone, 0 °C, 6 h; (d) BH3·THF, THF, 38 °C, 1 h; (e) PCC, CH2Cl2, rt, 2 h; (f) H2SO4, MeOH, 75 °C, 4 h; (g) FeCl3, CH2Cl2, rt, 5 h; (h) LiAlH4, THF, rt, 3 h; (i) Ph3P=CHCO2Et, toluene, reflux, 5 h; (j) KOH, EtOH/H2O (2:1), reflux, 5 h; (k) (i) (COCl)2, toluene, 70 °C, 14 h, (ii) NaN3, acetone, rt, 2 h, (iii) I2, 1, 2-dichlorbenzol, reflux, 2.5 h; (l) R-Br, NaH, DMF, 80 °C, 4 h; (m) LiAlH4, AlCl3, THF, rt, 4 h; (n) LiAlH4, THF, rt, 48 h, quench, MeOH/ H2O (100:1), rt. a

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CHEMISTRY

11. After experimentation, it was found that lithium aluminum hydride (LiAlH4) alone or in combination with aluminum chloride (AlCl3) was able to achieve this transformation. The use of LiAlH4 resulted in generation of products 10 (2−11% yields) and 11 (5−25% yields), whereas LiAlH4 in combination with AlCl3 greatly favored product 10 over 11 and thus generated high yields for 10 (42−95%) (see Supporting Information Table S1 and Experimental Section). The route used to synthesize 22a−22d is depicted in Scheme 2A. Nitrocinnamic acids (14) were synthesized through a Perkin reaction17 from commercially available phenylacetic

For the preparation of compounds 9−11, the procedure described in Chuang et al.16 was used for 9 and modified for 10 and 11 (Scheme 1). Increasing the size of the E ring was found to disfavor ring formation, but the synthesis of 9c was nevertheless achieved using the same route as for 7methoxycryptopleurine 9b, albeit in a lower yield. Reduction of 8 to give 9 was accomplished using sodium bis(2methoxyethoxy) aluminum hydride in dioxane, but these conditions were not effective in the reduction of 7 to 10 and C



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acids (12) and substituted o-nitrobenzaldehydes (13). Reduction of the nitro group of 14 afforded aminocinnamic acids (15). Phenanthrene acids (16) were obtained through a Pschorr cyclization17 from 15. Reduction of 16 with diborane in THF afforded 22a−22d.18 The route shown in Scheme 2B was followed as previously described19,20 to achieve the synthesis of 22e. Oxidation of phenanthrene alcohols (22a− 22e) with pyridinium chlorochromate in CH2Cl2 afforded the target intermediate phenanthrene aldehydes (23a−23e).21 Subsequently, aldehydes (23) were converted to isoquinolinones (28−33) with a modification of a procedure reported by Chuang et al.16 (Scheme 2C). Reduction of 27 with LiAlH4 in THF at room temperature for 48 h afforded dibenzoisoquinolines 28−32 in 13−29% and 33 in 14−31% yields, respectively. This reaction was also greatly improved by reducing 27 with LiAlH4 and AlCl3 (3:1 ratio) in THF at room temperature for 4 h to afford high yields of dibenzoquinolines 28−32 (40−80%). The position of hydroxylation was further confirmed by 2DNMR analyses (Figure 2).

hydrogen atom projection of the E ring optimized the potency of these compounds. Considering the N-alkyl substituent analogues formed by uncyclizing the tylophorine E ring, the propyl 10b analogue is the most potent compound when compared to ethyl, butyl, and pentyl analogues 10a, 10c, and 10d. When compared to the uncyclized B ring analogues,3 e.g., septicine (2 in Figure 1 and Table 1), the uncyclized E ring analogues retained most of the tylophorine activity (Tables 1 and 2), e.g., 2 and 10b compared to 9a. Of the branch analogues, in general, the branched methyl group at C1- or C2-position of alkyl substitution slightly or moderately increased the potency of these compounds. For example, isobutyl 10f and sec-butyl 10g were more potent than butyl 10c by ∼2−6-fold (Table 2). However, the introduction of polar hydroxyl and amine groups to the propyl alcohol 10′m and propylamine 10′n decreased their potency dramatically by ∼10−30-fold. Further replacement of heterocycle groups for the N-substituents, e.g., 3-cyclohexene (10h), 2-methyl-[1,3]dioxolane (10i), 2-ethyl-[1,3]dioxolane (10j), and 2-propoxytetrahydro-2H-pyranyl group (10k) also failed to improve potency compared to the alkyl-replaced analogues (Table 2). Further investigation of the methoxy substituents in the phenanthrene moiety of 10a and 10b was also carried out. Of these analogues, 29a and 29b, which were demethoxylated at position C2, were found to be more potent than 10a and 10b. Thus, the demethoxylation at C2 of phenanthrene moiety improved the activity of the uncyclized E-ring tylophorine analogues. However, the demethoxylation at the positions of 6 or 7 to give 30a, 30b, 31, and 32 dramatically weakened the potency. Methoxylation at C4 did not affect the potency significantly when comparing the activities of 28a to 10a, 28b to 10b, 28c to 10c, and 28d to 10e (Table 3). Further introduction of a hydroxyl group to C14 of the C4 demethoxylated compounds with a N-alkyl group (Table 4) increased the potency by ∼3−6-fold for 11a compared to 10a, by ∼9−20-fold for 33b compared to 29a, by ∼4−9-fold for 33c compared to 29b, and by ∼2-fold for 33d compared to 29c. However introduction of a hydroxyl group to the same position C14 of other compounds did not show same tendency for improving activity, e.g., no significant improvement observed for 33a compared to 28b; decreased potency occurred to 11b

Figure 2. Key COSY, NOESY, and HMBC correlations of 33a.

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RESULTS AND DISCUSSION Of the tylophorine cyclized E ring analogues synthesized, the quinolizidine 9b was the most potent inhibitor of cancer cell growth (Table 1), exerting greater activity than indolizidine 9a by ∼2-fold and than 9c by ∼6−8-fold. Similar results were reported for antofine cyclized E ring analogues. This result is consistent with that obtained from the corresponding antofine analogues reported.22 The same trends of potency for antiTGEV repliction in ST cells were also found for these compounds (Table 1). Conceivably, an appropriate size and

Table 1. The Effect of the E Ring Size and seco-Structure of Tylophorine Derivatives on the Anti-TGEV in ST Cells and Antiproliferative Activities against Carcinoma Cells

GI50 (μM)a

EC50 (μM)a

compd ID

NCI-H460

MCF7

SF268

HONE-1

NUGC-3

TGEV

9a 9b 9c 2b

0.23 0.10 0.93 17.0

0.24 0.11 0.96 18.5

0.27 0.14 0.96 24.2

0.27 0.14 1.15 17.3

0.19 0.14 0.62 14.5

0.08 0.03 1.05 15.4

GI50 and EC50 values expressed in μM as the mean values of at least three experiments each in duplicate. Values of SD were less than 30% of GI50 and EC50 values and data not shown. b(+)-S-Septicine isolated from Tylophora ovata.3 a

D



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Table 2. Anti-TGEV in ST Cells and Antiproliferative Activity against Carcinoma Cells of Tylophorine-Derived Dibenzoquinolines with a Variety of N-Substituents

GI50 (μM)a

compd

EC50 (μM)a

ID

R

NCI-H460

MCF7

HepG2

HONE-1

NUGC-3

A549

TGEV-IFA

10a 10b 10c 10d 10e 10f 10g 10h 10i 10j 10k 10l 10′m 10′n paclitaxelc

CH2CH3 (CH2)2CH3 (CH2)3CH3 (CH2)4CH3 CH(CH3)2 CH2CH(CH3)2 CH(CH3)CH2CH3 3-cyclohexene 2-methyl-[1,3]dioxolane 2-ethyl-[1,3]dioxolane (CH2)3OTHP (CH2)3NHBoc (CH2)3OH (CH2)3NH2

1.01 0.45 2.05 6.11 0.29 0.87 0.45 2.30 18.52 4.15 7.49 2.89 9.41 11.73 0.03

0.75 0.51 1.97 9.51 0.34 0.91 0.40 2.21 22.45 4.60 9.80 3.35 7.14 15.32 0.03

1.00 0.77 3.04 8.05 0.67 1.81 0.51 3.64 41.04 6.49 13.98 4.15 16.89 36.93 0.18

1.57 1.21 2.75 6.20 0.94 1.72 1.15 2.53 22.06 6.13 9.65 4.52 16.60 21.13 0.02

0.62 0.47 1.93 7.67 0.32 1.04 0.44 1.88 19.09 4.95 8.22 3.40 6.63 10.33 0.02

0.97 0.73 2.22 NDb 0.38 1.32 0.53 1.92 31.50 4.82 3.78 3.55 5.47 6.04 0.06

1.65 0.83 4.39 6.53 0.60 1.36 0.73 2.99 >50.00 31.89 30.84 1.027 19.28 23.42 NDb

GI50 and EC50 values expressed in μM as the mean values of at least three experiments each in duplicate. Values of SD were less than 30% of GI50 and EC50 values and data not shown. bND, not determined. cPaclitaxel, a positive control. a

Table 3. Anti-TGEV in ST Cells and Antiproliferative Activity against Carcinoma Cells of Tylophorine-Derived Dibenzoquinolines with a Variety of N-Substituents

GI50 (μM)a

substitution

EC50 (μM)a

compd ID

R1

R2

R3

R4

R5

NCI-H460

MCF7

HepG2

HONE-1

NUGC-3

A549

TGEV-IFA

28a 28b 28c 28d 29a 29b 29c 30a 30b 31 32

OCH3 OCH3 OCH3 OCH3 H H H OCH3 OCH3 OCH3 H

OCH3 OCH3 OCH3 OCH3 H H H H H H OCH3

OCH3 OCH3 OCH3 OCH3 OCH3 OCH3 OCH3 H H OCH3 H

OCH3 OCH3 OCH3 OCH3 OCH3 OCH3 OCH3 OCH3 OCH3 H OCH3

CH2CH3 (CH2)2CH3 (CH2)3CH3 CH(CH3)2 CH2CH3 (CH2)2CH3 (CH2)3CH3 CH2CH3 (CH2)2CH3 (CH2)2CH3 (CH2)2CH3

0.87 0.49 2.68 0.57 0.30 0.26 3.51 3.39 3.82 2.40 3.76

1.18 0.62 3.19 0.72 0.37 0.42 4.73 3.12 3.75 2.83 4.11

2.50 0.86 5.01 1.61 0.66 0.48 4.04 4.01 4.48 3.17 6.29

1.99 0.96 4.06 1.12 0.57 0.77 4.77 4.87 5.56 3.31 4.75

1.70 0.54 2.54 0.64 0.32 0.43 3.99 3.48 4.63 2.94 3.72

1.41 0.79 3.01 0.76 0.40 0.47 2.39 4.38 4.64 2.95 3.67

1.14 0.77 2.92 0.78 0.58 0.68 5.60 14.18 8.91 8.68 2.63

GI50 and EC50 values expressed in μM as the mean values of at least three experiments each in duplicate. Values of SD were less than 30% of GI50 and EC50 values and data not shown. a

bonding interaction in the target binding pocket, giving rise to the significantly improved potency. The dibenzoquinolines assayed above for anticancer cell growth also were tested for anti-TGEV (Tables 2−4) and antiinflammatory activity (Table 5) in vitro and found to exert the same trends of potency in these three different biological actvities. In addition, the cellular activities which were reported

and 11c compared to 10b and 10e, respectively, by a factor of ∼2−4. Thus, only the introduction of C14 hydroxyl group to 29a (a demethoxylated dibenzoquinoline at C2 and C4) synergized the effect of demethoxylation at C2 to give the most potent dibenzoquinoline 33b. Demethoxylation at C2 may allow the C14 hydroxyl group to participate in an important E



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Table 4. Anti-TGEV in ST Cells and Antiproliferative Activities against Carcinoma Cells of Tylophorine-Derived Dibenzoquinolines with a Hydroxyl Group at the C14 Position

GI50 (μM)a

compd

EC50 (μM)a

ID

R

NCI-H460

MCF7

HepG2

HONE-1

NUGC-3

A549

TGEV-IFA

11a 11b 11c 33a 33b 33c 33d

CH2CH3 (CH2)2CH3 CH(CH3) 2 (CH2)2CH3 CH2CH3 (CH2)2CH3 (CH2)3CH3

0.17 1.08 1.07 0.59 0.02 0.05 1.48

0.16 1.02 1.21 0.59 0.04 0.11 2.33

0.36 1.68 1.86 0.83 0.04 0.11 1.85

0.44 1.74 1.71 0.82 0.06 0.14 3.00

0.15 0.73 0.87 0.49 0.03 0.11 1.86

0.19 1.19 1.23 0.82 0.02 0.05 1.32

0.34 0.67 2.74 0.66 0.04 0.12 2.92

a GI50 and EC50 values expressed in μM as the mean values of at least three experiments each in duplicate. SD values were less than 30% of GI50 and EC50 and data not shown.

direct targets of these compounds and their fundamental mechanisms of action is under investigation in our laboratory. These compounds also potently inhibited the cytopathic effect induced by murine hepatitis virus in DBT cells (Figure 4 and Supporting Information Figure S2). Solubility testing found them to be ∼4−5-fold more soluble in DMSO (Table 6) and in DMA (data not shown), compared to their respective related pentacyclic compounds, e.g., 10b vs 9a and 10c vs 9b. For comparison of 33a to tylophorine 9a, ∼6-fold increase was found. This improvement gave the guide for the formula choice of in vivo tests followed. On the basis of these results, 10b, 29a, and 33b were selected for in vivo evaluation. Efficacy tests using paw edema and tumor xenograft murine models, as well as pharmacokinetic tests, were conducted. For the pharmacokinetic studies, compounds were administered to rats intravenously and orally each at 3 mg/kg body weight. Blood samples were taken and the plasma analyzed. It was found that 9a and 9b exhibited oral bioavailabilities of 66% and 53%, as described.8 Tylophovatine C isolated from T. ovata3 was tested and analyzed in parallel as a reference C14-hydroxytylophorine derivative. Pharmacokinetic parameters were also obtained (Table 7). Tylophovatine C, 10b, 29a, and 33b all exhibited good oral bioavailabilities of 54%, 63%, 105%, and 64%, respectively. In an A549 xenografted tumor mouse model, 33b significantly and effectively reduced the tumor volume at the dose of 10 mg/kg (oral administration) and resulted in a 61% volume reduction by the end of the test. This is the first report of an antitumor tylophorine-derived compound, orally active in vivo. There were no signs of overt toxicity during the course of the experiment (Figure 5A). In the rat paw edema model, intraperitoneal administration of 33b resulted in significant inhibition of acute inflammation by reducing the paw edema volume greater than 60% at doses of 3 and 5 mg/kg body weight, respectively (Figure 5B). The neurotoxicity of 33b was examined by a rota-rod test for motor coordination25 for three consecutive days. No adverse effect on motor coordination was observed from the 33b treated groups when compared to the control vehicle and nontumor-bearing groups. Nontheless, the group of 33b at the dose 10 mg/kg on the day 3 was found to have better coordination than those from the nontumor-

Table 5. Anti-inflammatory Activity of Tylophorine Derived Dibenzoquinolines in Terms of Suppression Nitric Oxide Production in LPS/IFNγ Stimulated RAW264.7 Cells Raw 264.7/LPS + IFNγ compd ID

EC50 (μM)a

CC50 (μM)a

9a 10a 10b 10e 11a 11b 11c 33b

0.27 1.42 1.38 0.86 0.38 2.20 2.05 0.07

1.24 >4.0 >4.0 >4.0 >2.0 >4.0 >4.0 0.24

a EC50 and CC50 values expressed in μM as the mean values of at least three experiments each in duplicate. SD values were less than 30% of EC 50 and CC50 and data not shown. EC50 was measured for the effective concentration for 50% inhibition of the compound treatment on the production of nitric oxide. CC50 was measured for the concentration of 50% inhibition of the compound treatment on the cell growth to distinguish the potency and cytotoxicity.

to account for modes of actions exerted by tylophorine in these three biological systems5,8,23,24 were also examined for these dibenzoquinolines. These compounds down-regulated the cyclin A2 expression and caused the accumulation of c-Jun in carcinoma cells (Figure 3A and Supporting Information S1A), inhibited iNOS and COX-2 protein expression in LPS/IFNγ stimulated RAW264.7 cells (Figure 3B and Supporting Information Figure S1B), and exerted anti-TGEV nucleocapsid (N) protein expression and TGEV induced apoptosis through inhibition of the activation of caspase 3 from cleavage of procaspase 3 in ST cells (Figure 3C,D and Supporting Information Figure S1C). Thus, these compounds likely retain the same modes of actions as tylophorine.5,8,23 In the three biological systems tested above, synthesis of RNA or protein are largely in demand, e.g., for cancer cell proliferation, viral replication, or production of proinflammatory factors. Common properties might be shared by the direct molecular targets of these compounds in the three biological systems, e.g., interfering in RNA or protein synthesis. Elucidation of the F



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Figure 3. Pharmacological activities of tylophorine and tylophorine-derived dibenzoquinolines in three biological systems. (A) Western analysis for expression of cyclin A2 and c-Jun in carcinoma cells after compound treatment for 24 h. Tylophorine compounds down-regulated cyclin A2 expression and induced c-Jun protein accumulation to exert their anticancer effect. (B) Western analysis for iNOS, COX-2, and GAPDH protein expression in LPS/INFγ stimulated RAW264.7 cells after compound treatment for 20 h. Tylophorine compounds inhibited the induction of iNOS and COX-2 expression in LPS/INFγ stimulated RAW264.7 cells to exert their anti-inflammatory effect. (C) Western analysis for TGEV nucleocapsid (N) protein, caspase 3, and GAPDH in TGEV infected ST cells at 16 hpi. Tylophorine compounds inhibited TGEV N protein expression and activation of caspase 3 through cleavage of pro-casapase 3 to exert their anti-TGEV effect. (D) Immunofluorescent assay for TGEV N and spike protein expression in TGEV infected ST cells at 6 hpi. Phase contrast images were shown for the field of ST cells assayed. (C) and (D) are for antiTGEV effect. The concentrations of treatment used for all the above experiments are 2 μM for 9a, 29a, and 11a, 4 μM for 10b and 10e, and 0.5 μM for 33b. The results shown are representative of at least three independent experiments.

bearing group with a *p value of 95% purity at 254 nm except 87.8% for 10′n, which could not be purified further (see Supporting Information Table S2). Solubility Determination in DMSO. Saturation concentrations for solubility of the test compounds in DMSO were determined by dissolving compounds in DMSO with assistance of sonication and the maximal volume before compound precipitation occurred was measured for calculation of solubility. General Procedure for the Preparation of 14a−c and 19. 6Nitroveratraldehyde (5 g, 23.7 mmol) and homoanisic acid (3.93 g, 23.7 mmol) were dissolved in acetic anhydride (20 mL), and then triethylamine (3.3 mL) was added slowly. The resultant solution was stirred at 90 °C for 15 h. At this point, H2O (2 mL) was added and the reaction was allowed to continue for 15 min. After, K2CO3 (26 g) in water (240 mL) was added slowly and the mixture was stirred for 60 °C for 1 h. The reaction mixture was cooled to 4 °C and acidified with 12N HCl. The aqueous solution was extracted with CH2Cl2, and the obtained organic phases were combined, washed with brine, dried, and then evaporated. Pure 14a (6.86 g) was obtained by further crystallization from EtOAc. General Procedure for the Preparation of 15a−c. A solution of 25% NH4OH (40 mL) was degassed with nitrogen for 30 min and heated to 100 °C before the addition of a FeSO4·7H2O (15 g, 56 mmol) with stirring. A solution of the nitrocinnamic acid 14a (2 g, 5.6 mmol) in 70 mL of 25% NH4OH was added slowly to the reaction mixture, under nitrogen at 100 °C for 2 h. After reaction was completed, the reaction mixture was cooled to room temperature to add decolorizing carbon and then the mixture was filtered. The filtrate was cooled to 4 °C, acidified to pH 3 with 85% H3PO4, and extracted with CHC13/2-propanol (3:1 v/v), washed with water, dried, and evaporated to give the aminocinnamic acid 15a (1.51 g). General Procedure for the Preparation of 16a−d. The aminocinnamic acid 15a (1.51 g, 4.6 mmol) was dissolved in acetone (250 mL) before subsequent slow additions of H2SO4 (0.5 mL, 9.2 mmol) and isoamyl nitrite (1.23 mL, 9.2 mmol) at 4 °C. In those cases where a precipitate was still present after 30 min of stirring, water was added until the solution became homogeneous. After 1 h, sodium iodide (3.6 g, 23.9 mmol) was added in five portions for 5 h. Sodium bisulfite was

added to turn the mixture yellow, whereupon it was poured into water (500 mL) and extracted with CHC13, washed with water, dried, and evaporated to give the phenanthrene acid 16a (1.07 g). General Procedure for the Preparation of 22a−d. The hydroboration solvent BH3·THF (1 M, 8.75 mL) was added, in three portions, to a stirred suspension of acid 16a (910 mg, 2.9 mmol) in THF (29 mL) over 1 h. Upon completion of addition, the reaction mixture was warmed (38 °C) for another hour, quenched (HOAc), and evaporated, and the residue was partitioned between 30 mL portions of CH2Cl2 and 10% NaOH. The organic layer was dried, filtered, and evaporated to give the alcohol 22a (778 mg). General Procedure for the Preparation of 20. Compound 19 (4.03 g, 12.8 mmol) was dissolved in 77 mL of MeOH containing 5.2 mL of concentrated H2SO4, and the mixture was refluxed for 4 h. The solvent was evaporated under reduced pressure, and the residue was partitioned between portions of CH2Cl2 and saturated NaHCO3 solution. The organic phase was dried, evaporated, and purified by silica gel column chromatography (EtOAc: hexane = 1:3 v/v) to give 20 (4.06 g). General Procedure for the Preparation of 21. Anhydrous FeCl3 (6.12 g, 37.8 mmol) was added to a solution of 20 (3.54 g, 10.8 mmol) dissolved in dry CH2Cl2 (108 mL) to react at room temperature under nitrogen for 20 h. The reaction solution was then evaporated under reduced pressure and the crude product purified by silica gel column chromatography (EtOAc:hexane = 1:3 v/v) to give 21 (1.88 g). General Procedure for the Preparation of 22e. A suspension of LiAlH4 (480 mg, 11.6 mmol) in 20 mL of dry THF was added to a solution of 21 (1.88 g, 5.8 mmol) dissolved in 30 mL of dry THF. The reaction mixture was stirred at room temperature under nitrogen for 3 h. The mixture was then quenched by 1.2 mL of H2O, 1.2 mL of 10% NaOH, and 2.4 mL of H2O, added sequentially. The mixture was filtered, and the filtrate was evaporated under reduced pressure. The residue was dissolved in CHC13, and the solution was washed with brine and dried over MgSO4. The combined organic phase was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (EtOAc:hexane = 1:1 v/v) to give alcohol 22e (1.52 g). I



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mixture was filtered, and the filtrate was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (MeOH:EtOAc:CH2Cl2 = 0:10:20, 1:10:20, 2:10:20; all solvent with 0.001% diethylamine) to afford the 29a and 33b mixtures. The mixtures were further purified by HPLC (5 μm, 10 mm × 250 mm; MeOH:EtOAc:CH2Cl2 = 1:10:20, 2:10:20; all solvent with 0.001% diethylamine) at 1 mL/min flow rate to afford 29a (15 mg) and 33b (31 mg). 2-Ethyl-6,7,10,11-tetramethoxy-1,2,3,4-tetrahydrodibenzo[f,h]isoquinoline (10a). Yield 9% (method e); white crystal; mp 186 °C. 1 H NMR (400 MHz, CDCl3): 1.31 (t, J = 7.2 Hz, 3H), 2.81 (quartet, J = 7.2 Hz, 2H), 2.96 (t, J = 5.6 Hz, 2H), 3.22 (t, J = 5.6 Hz, 2H), 4.02 (s, 2H), 4.04 (s, 3H), 4.05 (s, 3H), 4.12 (s, 6H), 7.15 (s, 1H), 7.29 (s, 1H), 7.82 (s, 1H), 7.83 (s, 1H). 13C NMR (75 MHz, CDCl3): 12.2, 26.7, 49.4, 52.0, 53.4, 55.8, 55.9, 56.9, 102.8, 103.3, 103.4, 103.8, 123.4, 123.5, 124.1, 124.7, 125.5, 125.6, 148.4, 148.5, 148.7. MS (EI) m/z 381 (M+, 100%). HRMS calcd for C23H27NO4 (M+) 381.1940; found 381.1930. 6,7,10,11-Tetramethoxy-2-propyl-1,2,3,4-tetrahydrodibenzo[f,h]isoquinoline (10b). Yield 76% (method d), 7% (method e); white crystal; mp 176−178 °C. 1H NMR (400 MHz, CDCl3): 1.02 (t, J = 7.6 Hz, 3H), 1.74 (sextet, J = 7.6 Hz, 2H), 2.67 (t, J = 7.6 Hz, 2H), 2.92 (t, J = 6.0 Hz, 2H), 3.18 (t, J = 6.0 Hz, 2H), 3.98 (s, 2H), 4.03 (s, 3H), 4.04 (s, 3H), 4.10 (s, 6H), 7.12 (s, 1H), 7.27 (s, 1H), 7.79 (s, 1H), 7.80 (s, 1H). 13C NMR (100 MHz, CDCl3): 12.0, 20.5, 27.2, 50.1, 54.3, 55.8, 55.9, 56.0, 60.6, 102.9, 103.2, 103.4, 103.8, 123.3, 123.4, 124.3, 125.6, 125.9, 148.3, 148.4, 148.6. MS (EI) m/z 395 (M+, 100%). HRMS calcd for C24H29NO4 (M+) 395.2097; found 395.2098. 2-Butyl-6,7,10,11-tetramethoxy-1,2,3,4-tetrahydrodibenzo[f,h]isoquinoline (10c). Yield 66% (method d); white needle; mp 172− 173 °C. 1H NMR (300 MHz, CDCl3): 1.00 (t, J = 7.5 Hz, 3H), 1.45 (sextet, J = 7.5 Hz, 2H), 1.70 (quintet, J = 7.5 Hz, 2H), 2.70 (t, J = 7.5 Hz, 2H), 2.91 (t, J = 5.7 Hz, 2H), 3.18 (t, J = 5.7 Hz, 2H), 3.97 (s, 2H), 4.03 (s, 3H), 4.04 (s, 3H), 4.10 (s, 6H), 7.12 (s, 1H), 7.26 (s, 1H), 7.79 (s, 1H), 7.80 (s, 1H). 13C NMR (75 MHz, CDCl3): 14.1, 20.8, 27.2, 29.4, 50.1, 54.3, 55.8, 55.9, 58.4, 102.8, 103.2, 103.3, 103.8, 123.3, 123.4, 124.2, 125.6, 125.9, 148.2, 148.3, 148.5, 148.6. MS (EI) m/z 409 (M+, 100%). HRMS calcd for C25H31NO4 (M+) 409.2253; found 409.2252. 6,7,10,11-Tetramethoxy-2-pentyl-1,2,3,4-tetrahydrodibenzo[f,h]isoquinoline (10d). Yield 5% (method e); white needle. 1H NMR (300 MHz, CDCl3): 0.95 (t, J = 7.2 Hz, 3H), 1.38−1.43 (m, 4H), 1.70−1.73 (m, 2H), 2.71 (t, J = 7.8 Hz, 2H), 2.94 (t, J = 5.4 Hz, 2H), 3.21 (t, J = 5.4 Hz, 2H), 4.01 (s, 2H), 4.04 (s, 3H), 4.05 (s, 3H), 4.12 (s, 6H), 7.16 (s, 1H), 7.30 (s, 1H), 7.83 (s, 1H), 7.84 (s, 1H). MS (APCI) m/z 424.2 [M + H]+. 2-Isopropyl-6,7,10,11-tetramethoxy-1,2,3,4-tetrahydrodibenzo[f,h]isoquinoline (10e). Yield 59% (method d), 2% (method e); white needle; mp 185 °C. 1H NMR (300 MHz, CDCl3): 1.26 (d, J = 6.6 Hz, 6H), 2.95 (t, J = 5.7 Hz, 2H), 3.09 (septet, J = 6.6 Hz, 1H), 3.17 (t, J = 5.7 Hz, 2H), 4.03 (s, 3H), 4.04 (s, 3H), 4.08 (s, 2H), 4.10 (s, 6H), 7.13 (s, 1H), 7.27 (s, 1H), 7.80 (s, 1H), 7.81 (s, 1H). 13C NMR (75 MHz, CDCl3): 18.5, 27.8, 45.1, 49.9, 54.1, 55.8, 55.9, 102.9, 103.2, 103.4, 103.8, 123.3, 124.4, 125.7, 126.1, 148.2, 148.3, 148.5. MS (EI) m/z 395 (M+, 100%). HRMS calcd for C24H29NO4 (M+) 395.2097; found 395.2103. 2-Isobutyl-6,7,10,11-tetramethoxy-1,2,3,4-tetrahydrodibenzo[f,h]isoquinoline (10f). Yield 69% (method d); light-yellow needle; mp 160−161 °C. 1H NMR (300 MHz, CDCl3): 1.02 (d, J = 6.6 Hz, 6H), 2.04 (septet, J = 6.9 Hz, 1H), 2.47 (d, J = 7.5 Hz, 2H), 2.90 (t, J = 6.0 Hz, 2H), 3.18 (t, J = 6.0 Hz, 2H), 3.97 (s, 2H), 4.03 (s, 3H), 4.04 (s, 3H), 4.11 (s, 6H), 7.13 (s, 1H), 7.29 (s, 1H), 7.80 (s, 1H), 7.81 (s, 1H). 13C NMR (75 MHz, CDCl3): 21.0, 25.8, 27.1, 50.2, 54.8, 55.8, 55.9, 56.0, 66.8, 102.9, 103.2, 103.4, 103.8, 123.3, 123.4, 124.3, 125.6, 125.8, 126.0, 148.3, 148.4, 148.6. MS (EI) m/z 409 (M+, 100%). HRMS calcd for C25H31NO4 (M+) 409.2253; found 409.2249. 2-(sec-Butyl)-6,7,10,11-tetramethoxy-1,2,3,4-tetrahydrodibenzo[f,h]isoquinoline (10g). Yield 95% (method d); light-yellow crystal; mp 157−158 °C. 1H NMR (300 MHz, CDCl3): 1.00 (t, J = 7.5 Hz, 3H), 1.19 (d, J = 6.3 Hz, 3H), 1.42−1.55 (m, 1H), 1.76−1.87 (m,

General Procedure for the Preparation of 23a−e. Phenanthrene alcohol 22a (835 mg, 2.8 mmol) dissolved in 75 mL of dry CH2Cl2 was added to a suspension of pyridinium chlorochromate (903 mg, 4.2 mmol) in 10 mL of dry CH2Cl2 at 0 °C. The reaction mixture was stirred at room temperature under nitrogen for 2 h. The mixture was diluted with 80 mL of ether and filtered. The solids were washed with CHC13, and the combined organic phase was concentrated by evaporation under reduced pressure. The residue was purified by silica gel column chromatography (CH2Cl2) to give the aldehyde 23a (726 mg). General Procedure for the Preparation of 24a−e. A mixture of 23a (0.73 g, 2.5 mmol) and (carboethoxymethylene)triphenylphosphorane (1.2 g, 3.4 mmol) in 20 mL of toluene was refluxed under nitrogen for 4 h. After cooling, the resulting solution was directly purified by silica gel column chromatography (CH2Cl2) to afford the ethyl ester 24a (0.89 g). General Procedure for the Preparation of 25a−e. A solution of 8 mL of 1 N KOH was added to a solution of the ester 24a (0.81 g, 2.2 mmol) in 16 mL of EtOH, and the reaction mixture was heated to reflux for 3 h. After cooling, the reaction solution was evaporated and the residue was dissolved in 16 mL water, acidified with 10% HCl, and extracted with 70 mL of CHCl3 and 60 mL of EtOAc. The combined extracts were dried with anhydrous MgSO4, filtered, and evaporated under reduced pressure to give the acrylic acid 25a (0.73 g) General Procedure for the Preparation of 26a−e. A mixture of 25a (1.26 g, 3.7 mmol) and oxalyl chloride (1.89 g, 14.8 mmol) in 63 mL of toluene was heated for 15 h at 70 °C. After cooling, the resulting mixture was concentrated under reduced pressure to afford the acyl chloride. The acyl chloride was added immediately to a suspension of NaN3 (0.73 g, 11.1 mmol) in 120 mL of dry acetone in an ice bath. The reaction mixture was stirred for 2 h at room temperature and filtered. The solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (CHCl3) to afford acryloyl azide. A mixture of azide and I2 (0.02 g, catalytic amount) in 18 mL of o-dichlorobenzene was refluxed for 2 h. After cooling, compound 26a was isolated by filtration and washed with CH2Cl2 to afford pure isoquinolinone 26a (1.01 g) General Procedure for the Preparation of 7a−l and 27a−k. A suspension of NaH (60% dispersion in oil, 119 mg, 2.98 mmol) in 1 mL of DMF cooled in an ice bath was added to a solution of 26a (400 mg, 1.2 mmol) in 15 mL of DMF with stirring at room temperature under nitrogen for 30 min. After the addition was completed, the mixture was added slowly to a solution of bromoethane (4.77 mmol) in 1 mL of DMF. The mixture was stirred at 80 °C for 4 h. The solvent was evaporated under reduced pressure, and water was then added. The mixture was extracted with CH2Cl2, dried over MgSO4, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (EtOAc:CH2Cl2 = 1:8 v/v) to afford the alkyll isoquinolinone of 27a (400 mg). General Procedure for the Preparation of 10a−n, 11a−c, 28a− d, 29a−c, 30a−b, 31, 32, and 33a−d. 1. Reduction by LiAlH4 and AlCl3 for Method d in Scheme 1 and Method m in Scheme 2. A solution of AlCl3 (18 mg, 0.13 mmol) in 0.5 mL of dry THF was added to a stirred suspension of 27a (48 mg, 0.13 mmol) in 2 mL of dry THF cooled at −15 °C. The mixture was stirred at room temperature under nitrogen for 15 min, and then LiAlH4 (26 mg, 0.65 mmol) was added at −15 °C. The reaction mixture was warmed up to room temperature for 4 h and then quenched by 5 mL of MeOH, and 5 mL of 10% NaOH sequentially. The mixture was filtered, and the filtrate was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (MeOH:EtOAc:CH2Cl2 = 1:10:20 v/v) to afford the dibenzo[f,h]isoquinolinone of 29a (19 mg). 2. Reduction by LiAlH4 for Method e in Scheme 1 and Method n in Scheme 2. A solution of LiAlH4 (136 mg, 3.58 mmol) in 5 mL of dry THF was added to a stirred suspension of 27a (100 mg, 0.28 mmol) in 8 mL of dry THF cooled at −15 °C,. The reaction mixture was warmed up to room temperature for 2 days, then 5.5 mL of MeOH/H2O (100:1) was added slowly using a syringe pump. The J



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2-(6,7,10,11-Tetramethoxy-3,4-dihydrodibenzo[f,h]isoquinolin2(1H)-yl)ethanamine (10′n). Yield 71% (method d); colorless rock; mp 161−162 °C. 1H NMR (300 MHz, CDCl3): 2.80 (t, J = 6.0 Hz, 2H), 2.94 (t, J = 6.0 Hz, 2H), 2.99 (t, J = 6.0 Hz, 2H), 3.19 (t, J = 5.7 Hz, 2H), 4.02 (s, 2H), 4.04 (s, 6H), 4.11 (s, 6H), 7.13 (s, 1H), 7.28 (s, 1H), 7.81 (s, 1H), 7.82 (s, 1H). MS (EI) m/z 396 (M+, 9%) and 349 (100%). HRMS calcd for C23H28N2O4 (M+) 396.2049; found 396.2043. 2-Ethyl-6,7,10,11-tetramethoxy-1,2,3,4-tetrahydrodibenzo[f,h]isoquinolin-4-ol (11a). Yield 25% (method e); white needle; mp 185 °C. 1H NMR (300 MHz, CDCl3): 1.25 (t, J = 7.2 Hz, 3H), 2.26 (d, J = 11.1 Hz, 1H), 2.47−2.58 (m, 1H), 2.68−2.79 (m, 1H), 3.00 (d, J = 15.6 Hz, 1H), 3.30−3.36 (m, 1H), 3.35 (d, J = 11.1 Hz, 1H), 3.84 (s, 3H), 4.07 (s, 3H), 4.09 (s, 3H), 4.12 (s, 3H), 4.91 (s, 1H), 6.25 (s, 1H), 7.40 (s, 1H), 7.55 (s, 1H), 7.79 (s, 1H). 13C NMR (100 MHz, CDCl3): 11.3, 52.2, 53.3, 55.6, 55.7, 55.8, 57.2, 64.6, 102.5, 102.5, 102.6, 105.1, 122.6, 123.6, 123.9, 125.3, 126.4, 126.6, 147.9, 148.2, 148.4, 148.5. MS (EI) m/z 397 (M+, 47%) and 340 (100%). HRMS calcd for C23H27NO5 (M+) 397.1889; found 397.1880. 6,7,10,11-Tetramethoxy-2-propyl-1,2,3,4-tetrahydrodibenzo[f,h]isoquinolin-4-ol (11b). Yield 14% (method e); white rock; mp 202 °C. 1H NMR (400 MHz, CDCl3): 1.02 (t, J = 7.2 Hz, 3H), 1.70−1.81 (m, 2H), 2.46 (d, J = 11.6 Hz, 1H), 2.52−2.59 (m, 1H), 2.65−2.71 (m, 1H), 3.28 (d, J = 15.2 Hz, 1H), 3.35 (d, J = 11.6 Hz, 1H), 3.80 (d, J = 15.2 Hz, 1H), 3.95 (s, 3H), 4.08 (s, 3H), 4.11 (s, 3H), 4.13 (s, 3H), 5.03 (s, 1H), 6.67 (s, 1H), 7.60 (s, 1H), 7.68 (s, 1H), 7.76 (s, 1H). 13C NMR (150 MHz, CDCl3): 12.0, 19.7, 54.0, 55.8, 55.9, 56.0, 58.0, 60.4, 64.7, 102.9, 103.0, 104.9, 123.0, 123.8, 124.2, 125.3, 126.7, 127.3, 148.3, 148.5, 148.7, 148.8. MS (EI) m/z 411 (M+, 50%) and 340 (100%). HRMS calcd for C24H29NO5 (M+) 411.2046; found 411.2049. 2-Isopropyl-6,7,10,11-tetramethoxy-1,2,3,4-tetrahydrodibenzo[f,h]isoquinolin-4-ol (11c). Yield 5% (method e); yellow crystal; mp 170−172 °C. 1H NMR (300 MHz, CDCl3): 1.24−1.28 (m, 6H), 2.68 (d, J = 11.7 Hz, 1H), 3.11−3.17 (m, 1H), 3.31 (d, J = 11.7 Hz, 1H), 3.89 (d, J = 15.3 Hz, 1H), 4.04 (s, 3H), 4.05 (s, 3H), 4.07 (s, 3H), 4.08 (s, 3H), 4.23 (d, J = 15.3 Hz, 1H), 5.14 (s, 1H), 7.12 (s, 1H), 7.70 (s, 1H), 7.79 (s, 1H), 7.81 (s, 1H). 13C NMR (150 MHz, CDCl3): 17.4, 19.5, 50.3, 52.8, 53.9, 55.9, 56.0, 56.1, 64.6, 103.1, 103.2, 103.3, 104.6, 123.6, 123.9, 124.4, 125.4, 127.4, 128.2, 148.6, 148.7, 149.0, 149.1. MS (ESI) m/z 412 (M + H)+. 2-Ethyl-6,7,8,10,11-pentamethoxy-1,2,3,4-tetrahydrodibenzo[f,h]isoquinoline (28a). Yield = 29% (method n); yellow rock; mp 199−200 °C. 1H NMR (400 MHz, CDCl3): 1.31 (t, J = 7.2 Hz, 3H), 2.79 (q, J = 7.2 Hz, 2H), 2.93 (t, J = 5.6 Hz, 2H), 3.19 (t, J = 5.6 Hz, 2H), 3.98 (s, 3H), 4.01 (s, 2H), 4.02 (s, 3H), 4.04 (s, 3H), 4.05 (s, 3H), 4.09 (s, 3H), 7.16 (s, 1H), 7.18 (s, 1H), 9.20 (s, 1H). 13C NMR (150 MHz, CDCl3): 10.1, 22.7, 46.9, 49.4, 50.6, 55.7, 55.8, 56.0, 60.6, 61.4, 100.3, 101.7, 108.3, 118.6, 123.1, 123.4, 124.1, 127.0, 143.1, 148.5, 148.8, 151.8, 152.2. MS (EI) m/z 411 (M+, 100%). HRMS calcd for C24H29NO5 (M+) 411.2046; found 411.2050. 6,7,8,10,11-Pentamethoxy-2-propyl-1,2,3,4-tetrahydrodibenzo[f,h]isoquinoline (28b). Yield 17% (method n); light-yellow crystal; mp 124−125 °C. 1H NMR (300 MHz, CDCl3): 1.03 (t, J = 7.5 Hz, 3H), 1.74 (sextet, J = 7.5 Hz, 2H), 2.68 (t, J = 7.5 Hz, 2H), 2.92 (t, J = 6.0 Hz, 2H), 3.18 (t, J = 6.0 Hz, 2H), 3.98 (s, 3H), 4.00 (s, 2H), 4.02 (s, 3H), 4.04 (s, 3H), 4.05 (s, 3H), 4.09 (s, 3H), 7.16 (s, 1H), 7.18 (s, 1H), 9.19 (s, 1H). 13C NMR (150 MHz, CDCl3): 12.0, 20.2, 27.1, 49.8, 54.0, 55.7, 55.8, 60.0, 60.5, 61.3, 100.4, 102.2, 108.1, 117.8, 123.5, 124.8, 125.6, 126.6, 128.4, 142.1, 148.0, 151.6, 151.7. MS (EI) m/z 425 (M+, 100%). HRMS calcd for C25H31NO5 (M+) 425.2202; found 425.2195. 2-Butyl-6,7,8,10,11-pentamethoxy-1,2,3,4-tetrahydrodibenzo[f,h]isoquinoline (28c). Yield 16% (method n); light-yellow crystal; mp 120−123 °C. 1H NMR (400 MHz, CDCl3): 1.00 (t, J = 7.6 Hz, 3H), 1.45 (sextet, J = 7.6 Hz, 2H), 1.70 (quintet, J = 7.6 Hz, 2H), 2.71 (t, J = 7.6 Hz, 2H), 2.92 (t, J = 5.6 Hz, 2H), 3.18 (t, J = 5.6 Hz, 2H), 3.98 (s, 3H), 4.00 (s, 2H), 4.02 (s, 3H), 4.05 (s, 3H), 4.06 (s, 3H), 4.09 (s, 3H), 7.16 (s, 1H), 7.18 (s, 1H), 9.19 (s, 1H). MS (ESI) m/z 440 (M + H)+.

2H), 2.81−2.90 (m, 1H), 2.93−3.01 (m, 1H), 3.15 (t, J = 5.4 Hz, 2H), 4.03 (s, 3H), 4.04 (s, 3H), 4.08 (s, 2H), 4.10 (s, 6H), 7.13 (s, 1H), 7.27 (s, 1H), 7.78 (s, 1H), 7.80 (s, 1H). 13C NMR (75 MHz, CDCl3): 11.5, 13.8, 26.3, 28.0, 29.6, 44.8, 49.8, 55.8, 55.9, 60.4, 102.9, 103.2, 103.3, 103.8, 123.3, 124.4, 125.8, 126.3, 126.5, 148.2, 148.3, 148.5. MS (EI) m/z 409 (M+, 31%) and 380 (100%). HRMS calcd for C25H31NO4 (M+) 409.2253; found 409.2257. 2-(Cyclohex-2-en-1-yl)-6,7,10,11-tetramethoxy-1,2,3,4tetrahydrodibenzo[f,h]isoquinoline (10h). Yield 70% (method d); light-yellow crystal; mp 128−129 °C. 1H NMR (400 MHz, CDCl3): 1.62−1.71 (m, 1H), 1.75−1.77 (m, 1H), 1.92−1.94 (m, 1H), 2.02− 2.03 (m, 1H), 2.04−2.08 (m, 2H), 2.91−2.96 (m, 1H), 3.04−3.09 (m, 1H), 3.17 (t, J = 5.2 Hz, 2H), 3.63 (br s, 1H), 4.03 (s, 3H), 4.04 (s, 3H), 4.10 (s, 6H), 4.16 (d, J = 6.0 Hz, 2H), 5.86 (d, J = 10.4 Hz, 1H), 5.95 (d, J = 10.4 Hz, 1H), 7.12 (s, 1H), 7.27 (s, 1H), 7.80 (s, 1H), 7.81 (s, 1H). 13C NMR (100 MHz, CDCl3): 21.7, 23.2, 25.4, 28.0, 45.2, 49.9, 55.8, 56.0, 60.0, 103.0, 103.2, 103.4, 103.9, 123.3, 123.4, 124.4, 125.8, 126.2, 126.3, 129.2, 130.6, 148.3, 148.4, 148.6. MS (EI) m/z 433 (M+, 100%). HRMS calcd for C27H31NO4 (M+) 433.2253; found 433.2259. 2-((1,3-Dioxolan-2-yl)methyl)-6,7,10,11-tetramethoxy-1,2,3,4tetrahydrodibenzo[f,h]isoquinoline (10i). Yield 42% (method d); white needle; mp 182−183 °C. 1H NMR (300 MHz, CDCl3): 2.96 (d, J = 4.2 Hz, 2H), 3.07 (t, J = 5.1 Hz, 2H), 3.19 (t, J = 5.1 Hz, 2H), 3.91 (AA′BB′, 2H), 4.02 (s, 3H), 4.03 (s, 3H), 4.04 (AA′BB′, 2H), 4.10 (s, 6H), 4.12 (s, 2H), 5.21 (t, J = 4.5 Hz, 1H), 7.12 (s, 1H), 7.26 (s, 1H), 7.79 (s, 1H), 7.80 (s, 1H). 13C NMR (75 MHz, CDCl3): 26.9, 51.0, 54.7, 55.8, 55.9, 60.9, 64.9, 102.9, 103.2, 103.3, 103.8, 123.3, 123.4, 124.2, 125.5, 125.7, 148.3, 148.4, 148.6. MS (EI) m/z 439 (M+, 13%) and 366 (100%). HRMS calcd for C25H29NO6 (M+) 439.1995; found 439.1987. 2-(2-(1,3-Dioxolan-2-yl)ethyl)-6,7,10,11-tetramethoxy-1,2,3,4tetrahydrodibenzo[f,h]isoquinoline (10j). Yield 45% (method d); white crystal; mp 186−187 °C. 1H NMR (300 MHz, CDCl3): 2.10 (td, J = 7.5, 4.5 Hz, 2H), 2.87 (t, J = 7.5 Hz, 2H), 2.94 (t, J = 5.7 Hz, 2H), 3.18 (t, J = 5.7 Hz, 2H), 3.88 (AA′BB′, 2H), 3.99 (s, 2H), 4.02 (AA′BB′, 2H), 4.03 (s, 6H), 4.10 (s, 6H), 5.04 (t, J = 4.8 Hz, 1H), 7.10 (s, 1H), 7.26 (s, 1H), 7.79 (s, 1H), 7.80 (s, 1H). 13C NMR (75 MHz, CDCl3): 27.2, 31.7, 50.1, 53.3, 54.2, 55.8, 55.9, 56.0, 64.9, 102.8, 102.9, 103.2, 103.3, 103.4, 103.8, 123.3, 123.4, 124.2, 125.4, 125.5, 125.8, 148.3, 148.4, 148.6. MS (EI) m/z 453 (M+, 51%) and 324 (100%). HRMS calcd for C26H31NO6 (M+) 453.2151; found 453.2141. 6,7,10,11-Tetramethoxy-2-(3-((tetrahydro-2H-pyran-2-yl)oxy)propyl)-1,2,3,4-tetrahydrodibenzo[f,h]isoquinoline (10k). Yield 7% (method e); yellow crystal. 1H NMR (400 MHz, CDCl3): 1.46−1.55 (m, 4H), 1.65−1.70 (m, 1H), 1.75−1.81 (m, 1H), 1.97 (quintet, J = 7.2 Hz, 2H), 2.73−2.77 (m, 2H), 2.87 (t, J = 5.6 Hz, 2H), 3.12 (t, J = 5.6 Hz, 2H), 3.41−3.52 (m, 2H), 3.81−3.86 (m, 2H), 3.93 (s, 2H), 3.96 (s, 3H), 3.97 (s, 3H), 4.00 (s, 6H), 4.53−4.55 (m, 1H), 7.06 (s, 1H), 7.20 (s, 1H), 7.73 (s, 1H), 7.74 (s, 1H). MS (EI) m/z 495 (M+, 9%) and 410 (100%). HRMS calcd for C29H37NO6 (M+) 495.2621; found 495.2627. tert-Butyl (2-(6,7,10,11-Tetramemthoxy-3,4-dihydrodibenzo[f,h]isoquinolin-2(1H)-yl)ethyl) carbamate (10l). Yield 47% (method d); yellow crystal; mp 123−124 °C. 1H NMR (300 MHz, CDCl3): 1.43 (s, 9H), 2.84 (t, J = 5.7 Hz, 2H), 2.94 (t, J = 5.7 Hz, 2H), 3.19 (t, J = 5.4 Hz, 2H), 3.44 (quartet, J = 5.7 Hz, 2H), 4.00 (s, 2H), 4.04 (s, 6H), 4.11 (s, 6H), 5.20 (br s, 1H), 7.10 (s, 1H), 7.28 (s, 1H), 7.80 (s, 1H), 7.81 (s, 1H). 13C NMR (75 MHz, CDCl3): 27.2, 28.4, 49.9, 54.0, 54.8, 55.8, 55.9, 56.0, 57.1, 102.8, 103.3, 103.4, 103.8, 123.4, 123.5, 124.1, 125.3, 125.5, 125.9, 148.4, 148.6, 148.7. MS (ESI) m/z 497 (M + H)+. 3-(6,7,10,11-Tetramethoxy-3,4-dihydrodibenzo[f,h]isoquinolin2(1H)-yl)propan-1-ol (10′m). Yield 93% (method d); white needle. 1 H NMR (300 MHz, CDCl3): 1.95 (quintet, J = 5.6 Hz, 2H), 2.99− 3.07 (m, 4H), 3.23 (t, J = 5.6 Hz, 2H), 3.90 (t, J = 5.6 Hz, 2H), 4.04 (s, 3H), 4.06 (s, 3H), 4.11 (s, 2H), 4.12 (s, 6H), 7.13 (s, 1H), 7.28 (s, 1H), 7.83 (s, 1H), 7.84 (s, 1H). MS (EI) m/z 411 (M+, 97%) and 349 (100%). HRMS calcd for C24H29NO5 (M+) 411.2046; found 411.2043. K



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2-Isopropyl-6,7,8,10,11-pentamethoxy-1,2,3,4tetrahydrodibenzo[f,h]isoquinoline (28d). Yield 13% (method n); light-yellow crystal; mp 195−198 °C. 1H NMR (300 MHz, CDCl3): 1.12 (d, J = 6.3 Hz, 6H), 2.97 (t, J = 5.4 Hz, 2H), 3.11 (septet, J = 6.3 Hz, 1H), 3.18 (t, J = 5.4 Hz, 2H), 3.98 (s, 3H), 4.02 (s, 3H), 4.04 (s, 3H), 4.06 (s, 3H), 4.09 (s, 3H), 4.11 (s, 2H), 7.16 (s, 1H), 7.18 (s, 1H), 9.19 (s, 1H). MS (ESI) m/z 426 (M + H)+. 2-Ethyl-7,10,11-trimethoxy-1,2,3,4-tetrahydrodibenzo[f,h]isoquinoline (29a). Yield 40% (method m), 16% (method n); yellow crystal; mp 160−161 °C. 1H NMR (300 MHz, CDCl3): 1.32 (t, J = 7.2 Hz, 3H), 2.80 (q, J = 7.2 Hz, 2H), 2.94 (t, J = 5.7 Hz, 2H), 3.25 (t, J = 5.7 Hz, 2H), 4.00 (s, 2H), 4.01 (s, 3H), 4.05 (s, 3H), 4.10 (s, 3H), 7.14 (s, 1H), 7.21 (dd, J = 9.0, 2.6 Hz, 1H), 7.89 (d, J = 2.6 Hz, 1H), 7.90 (s, 1H), 7.92 (d, J = 9.0 Hz, 1H). 13C NMR (150 MHz, CDCl3): 12.0, 20.4, 50.0, 54.0, 55.5, 55.9, 56.0, 60.3, 102.9, 104.0, 104.6, 114.8, 123.3, 125.1, 125.3, 125.5, 126.5, 127.4, 130.2, 148.2, 149.4, 157.6. MS (EI) m/z 351 (M+, 100%). HRMS calcd for C22H25NO3 (M+) 351.1834; found 351.1837. 7,10,11-Trimethoxy-2-propyl-1,2,3,4-tetrahydrodibenzo[f,h]isoquinoline (29b). Yield 13% (method n); yellow needle; mp 159− 160 °C. 1H NMR (400 MHz, CDCl3): 1.03 (t, J = 7.6 Hz, 3H), 1.75 (sextet, J = 7.6 Hz, 2H), 2.67 (t, J = 7.6 Hz, 2H), 2.92 (t, J = 6.0 Hz, 2H), 3.24 (t, J = 6.0 Hz, 2H), 3.98 (s, 2H), 4.02 (s, 3H), 4.05 (s, 3H), 4.11 (s, 3H), 7.15 (s, 1H), 7.21 (dd, J = 9.2, 2.4 Hz, 1H), 7.89 (d, J = 2.4 Hz, 1H), 7.91 (s, 1H), 7.93 (d, J = 9.2 Hz, 1H). 13C NMR (150 MHz, CDCl3): 12.0, 20.4, 26.9, 50.2, 53.9, 55.5, 55.8, 55.9, 60.5, 103.7, 103.8, 104.6, 114.8, 123.3, 123.9, 124.0, 125.0, 126.0, 126.8, 130.1, 148.3, 149.3, 157.4. MS (EI) m/z 365 (M+, 100%). HRMS calcd for C23H27NO3 (M+) 365.1991; found 365.1995. 2-Butyl-7,10,11-trimethoxy-1,2,3,4-tetrahydrodibenzo[f,h]isoquinoline (29c). Yield 15% (method n);yellow crystal; mp 130− 131 °C. 1H NMR (300 MHz, CDCl3): 1.00 (t, J = 7.5 Hz, 3H), 1.45 (sextet, J = 7.5 Hz, 2H), 1.71 (quintet, J = 7.5 Hz, 2H), 2.71 (t, J = 7.5 Hz, 2H), 2.92 (t, J = 5.7 Hz, 2H), 3.23 (t, J = 5.7 Hz, 2H), 3.98 (s, 2H), 4.02 (s, 3H), 4.05 (s, 3H), 4.11 (s, 3H), 7.12 (s, 1H), 7.21 (dd, J = 9.0, 2.6 Hz, 1H), 7.88 (d, J = 2.6 Hz, 1H), 7.90 (s, 1H), 7.92 (d, J = 9.0 Hz, 1H). MS (EI) m/z 379 (M+, 88%) and 336 (100%). HRMS calcd for C24H29NO3 (M+) 379.2147; found 379.2136. 2-Ethyl-6,7,11-trimethoxy-1,2,3,4-tetrahydrodibenzo[f,h]isoquinoline (30a). Yield 65% (method m); yellow crystal; mp 111− 112 °C. 1H NMR (300 MHz, CDCl3): 1.31 (t, J = 7.2 Hz, 3H), 2.79 (q, J = 7.2 Hz, 2H), 2.95 (t, J = 5.7 Hz, 2H), 3.23 (t, J = 5.7 Hz, 2H), 3.97 (s, 3H), 4.01 (s, 2H), 4.04 (s, 3H), 4.10 (s, 3H), 7.21 (dd, J = 8.9, 2.6 Hz, 1H), 7.24 (d, J = 2.6 Hz, 1H), 7.30 (s, 1H), 7.93 (s, 1H), 8.46 (d, J = 8.9 Hz, 1H). 13C NMR (150 MHz, CDCl3): 12.4, 27.3, 49.7, 52.3, 53.6, 55.4, 55.8, 55.9, 103.2, 103.8, 103.9, 115.0, 123.2, 124.1, 125.0, 125.5, 128.2, 130.6, 148.5, 148.7, 157.6. MS (EI) m/z 351 (M+, 100%). HRMS calcd for C22H25NO3 (M+) 351.1834; found 351.1836. 6,7,11-Trimethoxy-2-propyl-1,2,3,4-tetrahydrodibenzo[f,h]isoquinoline (30b). Yield 80% (method m); yellow crystal; mp 114− 115 °C. 1H NMR (300 MHz, CDCl3): 1.02 (t, J = 7.5 Hz, 3H), 1.75 (sextet, J = 7.5 Hz, 2H), 2.68 (t, J = 7.5 Hz, 2H), 2.94 (t, J = 6.0 Hz, 2H), 3.22 (t, J = 6.0 Hz, 2H), 3.97 (s, 3H), 4.00 (s, 2H), 4.04 (s, 3H), 4.10 (s, 3H), 7.21 (dd, J = 8.7, 2.7 Hz, 1H), 7.24 (d, J = 2.7 Hz, 1H), 7.30 (s, 1H), 7.93 (s, 1H), 8.46 (d, J = 8.7 Hz, 1H). 13C NMR (150 MHz, CDCl3): 11.9, 20.0, 26.5, 49.6, 53.4, 55.5, 55.8, 55.9, 59.9, 103.1, 103.5, 103.8, 115.3, 123.2, 124.1, 124.2, 124.8, 127.8, 130.3, 148.6, 148.8, 157.7. MS (EI) m/z 365 (M+, 90%) and 336 (100%). HRMS calcd for C23H27NO3 (M+) 365.1991; found 365.1987. 6,7,10-Trimethoxy-2-propyl-1,2,3,4-tetrahydrodibenzo[f,h]isoquinoline (31). Yield 60% (method m); yellow needle; mp 159− 160 °C. 1H NMR (400 MHz, CDCl3): 1.02 (t, J = 7.6 Hz, 3H), 1.74 (sextet, J = 7.6 Hz, 2H), 2.65 (t, J = 7.6 Hz, 2H), 2.93 (t, J = 6.0 Hz, 2H), 3.19 (t, J = 5.6 Hz, 2H), 4.02 (s, 3H), 4.05 (s, 5H), 4.11 (s, 3H), 7.20 (dd, J = 8.8, 2.4 Hz, 1H), 7.30 (s, 1H), 7.81 (d, J = 8.8 Hz, 1H), 7.90 (d, J = 2.4 Hz, 1H), 7.91 (s, 1H). MS (EI) m/z 365 (M+, 100%). HRMS calcd for C23H27NO3 (M+) 365.1991; found 365.1998. 7,8,11-Trimethoxy-2-propyl-1,2,3,4-tetrahydrodibenzo[f,h]isoquinoline (32). Yield 51% (method m); yellow crystal; mp 88−89 °C. 1H NMR (300 MHz, CDCl3): 1.01 (t, J = 7.5 Hz, 3H), 1.74 (sextet, J = 7.5 Hz, 2H), 2.66 (t, J = 7.8 Hz, 2H), 2.90 (t, J = 5.7 Hz,

2H), 3.21 (t, J = 5.7 Hz, 2H), 3.89 (s, 3H), 3.96 (s, 2H), 3.98 (s, 3H), 4.02 (s, 3H), 7.21 (d, J = 2.9 Hz, 1H), 7.24 (dd, J = 10.4, 2.9 Hz, 1H), 7.27 (d, J = 9.0 Hz, 1H), 7.75 (d, J = 9.0 Hz, 1H), 9.62 (d, J = 10.4 Hz, 1H). 13C NMR (150 MHz, CDCl3): 12.1, 20.5, 27.6, 50.2, 54.2, 55.3, 56.3, 59.7, 60.7, 104.2, 111.7, 113.8, 119.5, 122.8, 124.0, 125.9, 126.6, 128.8, 129.8, 132.9, 146.3, 151.0, 157.9. MS (EI) m/z 365 (M+, 86%) and 336 (100%). HRMS calcd for C23H27NO3 (M+) 365.1991; found 365.1987. 6,7,8,10,11-Pentamethoxy-2-propyl-1,2,3,4-tetrahydrodibenzo[f,h]isoquinolin-4-ol (33a). Yield 20% (method n), light-yellow crystal; mp 213−214 °C. 1H NMR (600 MHz, CDCl3): 1.01 (t, J = 7.8 Hz, 3H), 1.72 (sextet, J = 7.8 Hz, 2H), 2.54 (dd, J = 11.1, 2.1 Hz, 1H), 2.59 (ddt, J = 15.3, 12, 7.8 Hz, 1H), 2.68 (ddt, J = 15.3, 12, 7.8 Hz, 1H), 3.35 (d, J = 11.1 Hz, 1H), 3.47 (d, J = 15.3 Hz, 1H), 3.97 (s, 3H), 4.00 (s, 3H), 4.05 (s, 3H), 4.06 (s, 3H), 4.09 (s, 3H), 4.11 (d, J = 15.3 Hz, 1H), 5.09 (s, 1H), 6.96 (s, 1H), 7.64 (s, 1H), 9.16 (s, 1H). 13 C NMR (150 MHz, CDCl3): 11.9, 20.0, 54.4, 55.6, 55.7, 55.8, 57.9, 60.1, 60.5, 61.3, 64.9, 101.4, 102.5, 107.8, 118.2, 123.9, 124.4, 127.7, 128.1, 128.3, 142.2, 147.9, 148.5, 151.4, 151.9. MS (EI) m/z 441 (M+, 61%) and 370 (100%). HRMS calcd for C25H31NO6 (M+) 441.2151; found 441.2149. 2-Ethyl-7,10,11-trimethoxy-1,2,3,4-tetrahydrodibenzo[f,h]isoquinolin-4-ol (33b). Yield 31% (method n); yellow needle; mp 207−208 °C. 1H NMR (300 MHz, CDCl3): 1.28 (t, J = 7.2 Hz, 3H), 2.38 (dd, J = 11.4, 2.4 Hz, 1H), 2.60 (ddq, J = 14.7, 12.3, 7.2 Hz, 1H), 2.78 (ddq, J = 14.7, 12.3, 7.2 Hz, 1H), 3.18 (d, J = 14.7 Hz, 1H), 3.36 (d, J = 11.4 Hz, 1H), 3.62 (d, J = 14.7 Hz, 1H), 3.91 (s, 3H), 4.03 (s, 3H), 4.10 (s, 3H), 5.05 (s, 1H), 6.53 (s, 1H), 7.24 (dd, J = 9.3, 2.7 Hz, 1H), 7.64 (s, 1H), 7.72 (d, J = 2.7 Hz, 1H), 8.34 (d, J = 9.3 Hz, 1H). 13 C NMR (150 MHz, CDCl3): 12.1, 52.1, 53.7, 55.5, 55.8, 55.9, 57.3, 64.3, 103.0, 103.6, 104.5, 115.1, 124.1, 124.5, 124.7, 126.0, 126.2, 128.2, 130.6, 148.7, 149.2, 157.6. MS (EI) m/z 367 (M+, 42%) and 310 (100%). HRMS calcd for C22H25NO4 (M+) 367.1784; found 367.1780. 7,10,11-Trimethoxy-2-propyl-1,2,3,4-tetrahydrodibenzo[f,h]isoquinolin-4-ol (33c). Yield 16% (method n); yellow needle; mp 209−210 °C. 1H NMR (600 MHz, CDCl3): 1.01 (t, J = 7.2 Hz, 3H), 1.70−1.75 (m, 2H), 2.46 (d, J = 11.4 Hz, 1H), 2.51−2.56 (m, 1H), 2.63−2.68 (m, 1H), 3.29 (d, J = 14.4 Hz, 1H), 3.34 (d, J = 11.4 Hz, 1H), 3.81 (d, J = 14.4 Hz, 1H), 3.96 (s, 3H), 4.03 (s, 3H), 4.10 (s, 3H), 5.08 (s, 1H), 6.71 (s, 1H), 7.25 (dd, J = 9.0, 3.0 Hz, 1H), 7.72 (s, 1H), 7.78 (d, J = 1.8 Hz, 1H), 8.32 (d, J = 8.4 Hz, 1H). 13C NMR (150 MHz, CDCl3): 12.0, 19.7, 53.9, 55.5, 55.7, 55.8, 57.8, 60.4, 64.4, 102.9, 103.4, 104.3, 114.9, 124.0, 124.3, 124.7, 125.9, 126.3, 128.0, 130.5, 148.6, 149.0, 157.5. LRMS (EI+) m/z (rel intensity) 381 (M+, 42%) and 310 (100%). HRMS calcd for C23H27NO4 (M+) 381.1940; found, 381.1936. 2-Butyl-7,10,11-trimethoxy-1,2,3,4-tetrahydrodibenzo[f,h]isoquinolin-4-ol (33d). Yield 14% (method n); light-yellow crystal; mp 165−166 °C. 1H NMR (600 MHz, CDCl3): 1.01 (t, J = 7.2 Hz, 3H), 1.41−1.45 (m, 2H), 1.66−1.70 (m, 2H), 2.42 (d, J = 11.4 Hz, 1H), 2.50−2.55 (m, 1H), 2.64−2.69 (m, 1H), 3.22 (d, J = 15.6 Hz, 1H), 3.35 (d, J = 12.0 Hz, 1H), 3.67 (d, J = 14.4 Hz, 1H), 3.94 (s, 3H), 4.02 (s, 3H), 4.10 (s, 3H), 5.05 (s, 1H), 6.61 (s, 1H), 7.24 (dd, J = 9.0, 3.0 Hz, 1H), 7.68 (s, 1H), 7.75 (d, J = 1.8 Hz, 1H), 8.33 (d, J = 9.6 Hz, 1H). 13C NMR (150 MHz, CDCl3): 14.1, 20.8, 28.5, 53.9, 55.5, 55.7, 55.8, 57.8, 58.3, 64.3, 102.8, 103.3, 104.3, 114.9, 123.9, 124.2, 124.8, 125.9, 126.4, 127.9, 130.5, 148.5, 148.9, 157.5. LRMS (EI+) m/z (rel intensity) 395 (M+, 10%) and 310 (100%). HRMS calcd for C24H29NO4 (M+) 395.2097; found, 395.2098. Biology. Statistical Analysis. InStat version 3.06 software for Windows (GraphPad, San Diego, CA) was used to evaluate the statistical significance between two groups by 2-tailed unpaired Student’s t test. Throughout the text, figures, and legends, the following terminology is used to denote statistical significance: *p < 0.001; #p < 0.01; ns, no significance. Western Analysis. These assays were performed as described.5,23,26 Cyclin A2, c-Jun, iNOS, COX-2, and GAPDH proteins were analyzed by immunoblotting with anti-Cyclin A2, anti-c-Jun (Santa Cruz Biotechnology, Santa Cruz, CA), anti-iNOS (BIOMOL Research L



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Laboratories, Plymouth Meeting, PA), anti-COX-2 (Upstate Biotechnology), and anti-GAPDH (Cell Signaling, Beverly, MA) antibodies, respectively. In Vitro Carcinoma Cell Growth Inhibitory Assay. The previously described assay procedure was used.23,27 The human lung carcinoma cell line A549 cells (ATCC CCL-185) were grown in normal RPMI 1640 culture medium (GIBCO-Life Technologies, Inc., Gaithersburg, MD) supplemented with 2 g/L of NaHCO3, 2 mM L-glutamine, and 10% fetal bovine serum (FBS, Hyclone Laboratory Inc., Greeley, Co). NUGC-3 gastric carcinoma, HONE-1 nasopharyngeal carcinoma, MCF-7 breast carcinoma, NCI-H460 lung carcinoma, SF-268 glioblastoma, HepG2 hepato carcinoma, and A549 lung carcinoma cells were seeded at 4500, 6000, 6500, 2500, 7500, 10000, and 5000 cells/well, respectively, in 96-well plates. The positive control paclitaxel was purchased from Calbiochem-EMD Millipore (cat. 580555) with purity of ≥97% by HPLC. In Vitro Anti-inflammatory Effect by Determining Nitric Oxide Production. The previously described assay procedure was employed.5,28 RAW264.7 cells were seeded (65000 cells/well) and cultured in 96-well plates. After 24 h incubation, the medium was replaced with complete medium containing IFNγ (20 ng/mL)/LPS (5 μg/mL), and the test compounds were added at various concentrations as indicated for 18 h before measurement of nitric oxide production by the Nitrate/Nitrite assay kit as described. Immunofluorescent Assay (IFA). The IFA assay was performed and the images were captured as described.26 Briefly, ST cells in 96-well plates, with or without a 2 h pretreatment with test compounds, were infected with TGEV at a multiplicity of infection (MOI) of 10 for IFA. The IFA was performed at 6 h postinfection (hpi) with antibodies against the spike (S) and nucleocapsid (N) proteins of TGEV. The cells were treated with 10 different concentrations of test compounds. The results of these assays were used to obtain the dose−response curves from which 50% maximal effective concentration (EC50) values were determined. Cytopathic Effect Induced by Murine Hepatitis Virus in DBT Cells. The murine astrocytoma cell line DBT (a kind gift from Dr. Lai, Michael M. C.; Academia Sinica, Taiwan, R.O.C.)29 and embryonic murine hepatocyte cell line BNL CL.2 (BCRC no. 60180) was maintained as monolayer culture in Dulbecco's Modified Eagles Medium (DMEM, Hyclone Laboratory Inc., Greeley, Co) supplemented with 10% heat-inactivated FBS. The murine hepatitis virus (MHV), JHM strain (a gift from Dr. Jan, Jia-Tsrong; Academia Sinica, Taiwan, R.O.C.), was propagated in BNL CL.2 cells30 and harvested and the viral titer was measured. The DBT culture condition and the MOI of MHV-JHM used were optimized and determined in order to induce evident cytopathic effects at 24 hpi. DBT cells (2.8 × 105 cells/ well) were seeded onto the 24-well plates the day before infection with MHV-JHM. Prior to the addition of test compounds, the culture medium was changed to DMEM containing 2% FBS for MHV-JHM infection. The test compounds were pretreated for 1 h prior to MHVJHM infection at MOI of 0.005, and the cytopathic effects in DBT cells were observed at 24 hpi and recorded using a charge-coupled device linked to a Nikon Eclipse TE2000 microscopy. Subsequently, these MHV-infected DBT cells were subject to immunofluorescent assay as described above with a monoclonal antibody against MHV N protein generated in our laboratory (data not shown and will publish elsewhere). Animal Study Protocols. Animal study protocols [(NHRI-IACUC096006-A, 04/20/2007−04/19/2010), (NHRI-IACUC-096036-A, 11/01/2007−10/31/2010), (NHRI-IACUC-099025-A, 04/20/ 2010−04/19/2013), (NHRI-IACUC-099047-A, 10/31/2010−10/ 31/2013), (NHRI-IACUC-100112-A, 01/01/2012−12/31/2015), and (NHRI-IACUC-099065-A, 08-10-2010−08-09-2013)] were reviewed and approved for the in vivo experiments herein by the Institutional Animal Care and Use Committee of National Health Research Institutes, Taiwan. Pharmacokinetic Analysis. The Sprague−Dawley rats for the pharmacokinetic study were obtained from BioLASCO Taiwan Co. (Ilan, Taiwan) and housed in the animal facility at the National Health Research Institutes, Taiwan. The animal studies were performed

according to committee-approved procedures. Male rats (330−380 g, 9−10 weeks old) were quarantined for 1 week before use. The animals were surgically implanted with a jugular vein cannula 1 day before treatment and were fasted before treatment. Compounds 10b, 29a, and 33b at 3 mg/kg as well as tylophovatine C at 1.5 mg/kg were given to rats (n = 3) by intravenous or oral administration as prepared in a mixture of DMA/PEG400 (30/70, v/v) for 10b, 29a, and 33b and of DMA/PEG400 (50/50, v/v) for tylophovatine C. The volume of the dosing solution given was adjusted according to the body weight recorded before the drug was administered. At 0 (immediately before dosing), 2, 5 (intravenous only), 15, and 30 min and 1, 2, 4, 6, 8, 12, and 24 h after compound administration, a blood sample (150 μL) was taken from each animal via the jugular vein cannula and stored in ice (0−4 °C). The processing of the plasma and subsequent analysis by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS) were as described.31 The plasma concentration data were analyzed by a standard noncompartmental method with the Kinetica software (InnaPhase, Philadelphia, PA, USA). In Vivo Anti-inflammatory Activity Measured by CarrageenanInduced Hind-Paw Edema Test in Rats. Female Sprague−Dawley rats (193−214 g, 8−9 week old, n = 5) were used. To produce inflammation, 100 μL of 1% carrageenan solution in normal saline was injected into the left hind paw subplantar tissue. One hour before carrageenan challenge, the sample preparations (3 or 5 mg/kg) were injected intraperitoneally to the divided groups, with DMSO (100 μL) injected into the vehicle control group. The tests were performed as described.28 The positive control indomethacin was purchased from Sigma-Aldrich (cat. I7378) with purity of ≥99% (TLC). When inhibition is or greater than 30%, the treatment is considered with a significant efficacy. In Vivo Antitumor Activity. Four week-old male nude mice (NuFoxn1nu, BioLASCO Taiwan Co., Ltd.) were used. Nude mice were subcutaneously injected with 10 million A549 cells (in 200 μL of RPMI-1640 medium) into the right flank. When the average size of tumors reached ∼60 mm3, mice were randomly divided into three groups (n = 8 for treatments of vehicle and 33b at the dose of 10 mg/ kg; n = 7 for treatment of 33b at the dose of 5 mg/kg) and treated with vehicle control (25% DMA + 75% PEG400), 33b (5 mg/kg), and 33b (10 mg/kg) via oral gavage. Each group was administered once daily, on day 1−10, day 15−19, and day 22−26, for 20 times in total. The tumor volume was calculated using the equation V (mm3) = a × b2/2, where a is the largest diameter and b is the smallest diameter.32 Tumor growth inhibitions (TGI) were determined for antitumor effects which are expressed as % (Tday60 − Tday1)/Cday60 − Cday1) (T/C, treated versus control), dividing the tumor volumes from treatment groups with the control groups and multiplied by 100%. The effective criteria for the T/C (%) according to the National Cancer Institute standard is

Synthesis and Biological Evaluation of Tylophorine-Derived

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